Hepatocyte Based Insulin Gene Therapy For Diabetes

ABSTRACT

A method and vectors for controlling blood glucose levels in a mammal are disclosed. In one embodiment, the method comprises the steps of: treating the hepatocyte cells of a patient with a first, second or third vector, wherein the first vector comprises a promoter enhancer, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase and an albumin 3′UTR and lacks an HGH intron, wherein the second vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase site and an albumin 3′UTR and lacks a promoter enhancer, wherein the third vector comprises an HGH intron, glucose inducible regulatory elements, a liver-specific promoter, a gene encoding human insulin with modified peptidase site, an albumin 3′UTR and a promoter enhancer and observing the patient&#39;s insulin levels, wherein the patient&#39;s insulin levels are controlled.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 61/494,134, filed Jun. 7, 2011, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND

This invention relates to treatment of diabetes using hepatocyte-basedtherapy, and specifically to a method of utilizing hepatocyte cellscomprising a genetic construct that has a coding sequence for aproinsulin expressible in the cells in response to glucose levels. Theproinsulin synthesized in the cells is further processed into asecretable, active insulin.

Insulin is normally produced in and secreted by the beta cells of theislets of Langerhans in the pancreas. Mature insulin is a protein havingtwo polypeptide chains, A and B, held together by disulfide bonds. Theglucose responsive release of insulin from the beta cells is a complexevent including gene expression, posttranslational modification andsecretion. The initial protein product and insulin precursor ispreproinsulin, a single polypeptide chain having an N-terminal signalsequence and an intervening sequence, the C-peptide, between the B and Achains. The signal sequence is cleaved during transport from the roughendoplasmic reticulum to form proinsulin. The proinsulin is packagedinto secretory granules along with specific enzymes required for itsprocessing. Proinsulin folds into a specific three-dimensionalstructure, forming disulfide bonds. Mature insulin results from removalof the C-peptide. In beta cells, this function is catalyzed byendopeptidases that recognize the specific amino acid sequences at thejunction of the B chain and the C peptide (B-C junction) and at thejunction of the C chain and the A peptide (C-A junction). Matureinsulin, stored in secretory granules, is released in response toelevated blood glucose levels. The detailed mechanism of insulin releaseis not completely understood, but the process involves migration to andfusion of the secretory granules with the plasma membrane prior torelease.

In normally functioning beta cells, insulin production and release isaffected by the glycolytic flux. Glucokinase and glucose transporter 2(GLUT-2) are two proteins that are believed to be involved in sensingchanges in glucose concentration in beta cells. A reduction in GLUT-2,which is involved in glucose transport, is correlated with decreasedexpression of insulin; loss of glucokinase activity causes a rapidinhibition of insulin expression.

Autoimmune destruction of pancreatic beta cells causes insulin-dependentdiabetes mellitus or Type I diabetes. As a consequence of partial orcomplete loss of beta cells, little or no insulin is secreted by thepancreas. Most cells, with the exception of brain cells, require insulinfor the uptake of glucose. Inadequate insulin production causes reducedglucose uptake and elevated blood glucose levels. Both reduced glucoseuptake and high blood glucose levels are associated with a number ofvery serious health problems. In fact, without proper treatment,diabetes can be fatal.

One conventional treatment for diabetes involves periodic administrationof injectable exogenous insulin. This method has extended the lifeexpectancy of millions of people with the disease. However, bloodglucose levels must be carefully monitored to ensure that the individualreceives an appropriate amount of insulin. Too much insulin can causeblood glucose levels to drop to dangerously low levels. Too littleinsulin will result in elevated blood glucose levels. Even with carefulmonitoring of blood glucose levels, control of diet, and insulininjections, the health of the vast majority of individuals with diabetesis adversely impacted in some way. Replacement of beta cell function isa treatment modality that may have certain advantages over insulinadministration, because insulin would be secreted by cells in responseto glucose levels in the microenvironment. One way of replacing betacell function is by pancreas transplantation, which has met with somesuccess. However, the supply of donors is quite limited, and pancreastransplantation is very costly and too problematic to be made widelyavailable to those in need of beta cell function.

There have been many other proposed alternatives for beta cellreplacement, including replacing beta cell function with actual betacells or other insulin-secreting, pancreas-derived cell lines (Lacy, etal., Ann. Rev. Med., 37:33, 1986). Because the immune system recognizesheterologous cells as foreign, the cells have to be protected fromimmunoactive cells (e.g., T-cells and macrophages mediating cytolyticprocesses). One approach to protect heterologous cells is physicalimmunoisolation; however, immunoisolation itself poses significantproblems.

U.S. Pat. No. 5,427,940 issued to Newgard discloses another approach tobeta cell replacement. This patent describes an artificial beta cellproduced by engineering endocrine cells of the At-T-20 ACTH secretingcells. A stably transfected cell, At-T-20, is obtained by introducingcDNA encoding human insulin and the glucose transporter gene, i.e. theGLUT-2 gene, driven by the constitutive CMV promoter. The cell linealready expresses the correct isoform of glucokinase required forglucose responsive expression of the proinsulin gene. Although the cellline is responsive to glucose, it is secretagogue-regulated atconcentrations below the normal physiological range. Therefore, use ofthese cells in an animal would likely cause chronic hypoglycemia;furthermore, these cells are derived from a heterologous source and bearantigens foreign to the recipient host.

U.S. Pat. No. 5,534,404 issued to Laurance et al. discloses anotherapproach to obtaining a cell line in which insulin production issecretagogue-regulated. Subpopulations of beta-TC-6 cells having anincreased internal calcium concentration, a property associated withinsulin secretion, were selected using a cell sorter. After successivepassages, a subpopulation of cells that produce insulin in response toglucose in the physiological range (4-10 mM) was selected, and the cellswere encapsulated for therapeutic use in alginate bounded by a PAN/PVCpermselective hollow fiber membrane according to the method of Dionne(International Patent application No. PCT/US92/03327).

Valera, et al., FASEB Journal, 8: 440 (1994) describes transgenic mousehepatocytes expressing insulin under the control of the phosphoenolpuruvate carboxy kinase (PEPCK) promoter. The PEPCK promoter issensitive to the glucagon/insulin ratio and is activated at elevatedglucose levels. The PEPCK/insulin chimeric gene was injected intofertilized mouse eggs and offspring were screened for integration of thetransgene. In transgene positive mice, under conditions of severe isletdestruction by streptozotocin (SZ), the production and secretion ofintact insulin by the liver compensated for loss of islet function.Despite these prior art attempts, there is a continuing need foralternative methods to conventional insulin therapy for the treatment ofdiabetes.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method for obtainingglucose-regulated expression of insulin ex vivo in hepatocyte cells,wherein the method comprises delivering a first, second or third geneticvector for glucose-regulated synthesis of insulin into an isolatedhepatocyte cell wherein glucose-regulated expression of insulin occurs.The first vector comprises a promoter enhancer, 1-5 glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase sites and an albumin 3′UTR and lacks an HGHintron. The second vector comprises an HGH intron, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase sites and an albumin 3′UTR and lacks a promoterenhancer. The third vector comprises an HGH intron, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase sites, an albumin 3′UTR and a promoter enhancer.

In one embodiment, the invention comprises the step of transplanting thehepatocytes back into a mammal.

In one embodiment, the genetic vector is delivered by exposing the cellsto a virus infective for the cells, wherein the virus comprises thegenetic construct, and whereby at least a portion of the cells areinfected by the virus under suitable conditions and at a sufficientmultiplicity.

In one embodiment the mammal is human and the insulin is human insulin.

In another embodiment, the invention is a vector, as described above,suitable for controlling blood glucose levels.

In another embodiment, the invention is a method of controlling bloodglucose levels in a mammal, comprising the steps of treating a mammalwith a first, second or third vector, as described above.

In one embodiment of the invention, the mammal's cholesterol leveldecreases after treatment.

In one embodiment of the invention, the mammal's triglyceride leveldecreases after treatment.

The method of claim 10 wherein the mammal is a cat or dog.

Other embodiments of the present invention are described in thespecification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram comparing fasting blood glucose versus time inSTZ-treated diabetic rats treated with various levels of TA-1M.

FIG. 2 is a graph comparing postprandial blood glucose levels versustime for TA-1 minicircle DNA treated STZ-treated diabetic rats.

FIG. 3 is a graph comparing fasting body weight versus time in TA-1minicircle DNA treated STZ-treated diabetic rats.

FIG. 4 is a diagram of the TA1 expression cassette.

FIG. 5 is a diagram of the TA4 expression cassette.

FIG. 6 is the DNA sequence of the TA1 expression cassette.

FIG. 7 is the DNA sequence of the TA4 expression cassette.

FIG. 8 is a diagram of glucose levels versus time versus insulin levelsduring an intraperitoneal glucose tolerance test of TA1-treated diabeticrats.

FIG. 9 is a bar chart comparing ex vivo insulin production inhepatocytes treated with various insulin gene constructs.

FIG. 10 is a diagram of the TA2 expression cassette.

FIG. 11 is the DNA sequence of the TA2 expression cassette.

FIG. 12 is a diagram of blood glucose versus days in rats, fastedovernight and treated with TA1 and Ta4 constructs.

FIG. 13 is a diagram of body weight versus days in rats treated with TA1gene construct.

FIG. 14 is a graph of body weight versus days on rats treated with TA1and TA4 gene construct.

FIG. 15 is a table of human insulin levels in serum of diabetic ratstreated with plasmid or minicircle DNA.

FIG. 16 is a graph of blood glucose levels versus time for ratsexperiencing a second treatment of TA1 minicircle DNA.

FIG. 17 is a table of glucose-dependent insulin production from humanstem cells derived from hepatocytes.

FIG. 18 is a graph of body weight versus days for diabetic rats treatedwith TA1M, TA2M and TA3M.

DESCRIPTION OF THE INVENTION In General

Due to a shortage of donor pancreata and the limited long-term successof islet transplants, alternatives for treating Type I diabetes (T1D)are needed. We have developed a gene therapy-based glucose regulatedhepatic insulin production therapy that demonstrates great promise intreating T1D in experimental animals. Our approach is to apply insulingene therapy to autologous native hepatocytes or stem cell-derivedhepatocytes in an attempt to overcome the two critical shortcomings intreating T1D, which are the shortage of donor organs and the need forlife-long use of immunosuppression in transplantation patients.

As the Examples below demonstrate, we examined novel DNA constructs forthe ability to improve insulin production. For example, a novel insulinconstruct (TA1, described below) which contains the human growth hormone(HGH) intron, a translational enhancer, glucose inducible regulatoryelements, albumin promoter, human insulin with modified peptidase sites,and the albumin 3′-UTR improved insulin production in culturedhepatocytes and diabetic rats and mice. TA1 resulted in a ˜25-foldincrease in insulin production from isolated rat hepatocytes compared toour previously published insulin construct [Alam & Sollinger,Transplantation. 2002 Dec. 27; 74(12):1781-7].

In one aspect, the present invention is a novel DNA construct designedto improve insulin production in hepatocytes. Another aspect of thepresent invention is the creation of hepatocytes with improved insulinproduction. Another aspect of the present invention is a method ofrelieving the symptoms of Type I diabetes in a mammalian patient bymodulating the production of insulin.

Constructs of the Present Invention

The Examples below demonstrate four insulin constructions containingvarious elements. The Examples demonstrate that three constructs (TA1,TA2 and TA4) were successful in providing an insulin gene therapy thatprovides tight control of insulin production. Therefore, the presentinvention encompasses three types of vector. The first vector typecomprises a transcriptional enhancer, glucose inducible regulatoryelements, a gene promoter, a translational enhancer, a gene encodinginsulin with modified peptidase site and an albumin 3′ UTR and lacks anHGH intron. The second vector type comprises an HGH intron, glucoseinducible regulatory elements, a gene promoter, a translationalenhancer, a gene encoding insulin with modified peptidase site and analbumin 3′ UTR and lacks a transcriptional enhancer. The third vectortype comprises all of the listed elements.

In another embodiment of the present invention, the constructs of thepresent invention consist essentially of the elements listed above. By“consist essentially of” we mean that a vector of the present inventionwill consist of the element described above and possibly otherregulatory elements necessary for vector function. For example, plasmidsand minicircle vectors may include sequences to facilitate the additionor removal of functional elements, such as restriction sites, orsequences necessary for the replication of the vector itself.

Applicants note that SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 (FIGS.6, 7 and 11) are the entire nucleotide sequence of the TA1, TA2 and TA4expression constructs, respectively. These listings do not includesequences that correspond to minicircle recombination sites, etc. Forexample, SEQ ID NOs: 1 and 2 do not include specific sequences used forrecombination that are found within the commercial minicircle parentalplasmid that flank the expression cassettes. Because all these sequencesare part of commercial plasmids and readily available, the sequences arenot included in the provided information.

SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:3 include elements that arenecessary for the present invention (for example, the elements listedbelow) and linking and nonessential sequences that are useful forcloning but may be substituted by many other sequences with similarfunctions.

Residue Residue Residue Location Location Location in SEQ ID in SEQ IDin SEQ ID NO: 1 (TA1) NO: 2 (TA4) NO: 3 (TA2) Alpha fetoprotein (AFP) 15-265 —  15-265 Enhancer Glucose Inducible 279-369  46-136 279-369Regulatory Elements Albumin Promoter 380-706 147-474 380-707 SequenceHGH Intron — 475-754 708-987 VEGF Translational 707-870 755-918 988-1151 Enhancer Gene Encoding Human  871-1203  919-1251 1152-1484Insulin with Modified B-C and C-A Peptide Junctions for FurinCompatibility Albumin 3′ UTR 1204-2077 1252-2125 1485-2358

More specifically, the vectors of the present invention comprise thefollowing elements:

Promoter Enhancer

By “promoter enhancer,” we preferably mean the alpha-fetoproteinenhancer. The Examples below disclose the use of the alpha-fetoproteinenhancer. This element is designed to enhance transcription of thefunctionally linked gene sequence encoding a protein in liver cells.Alpha-fetoprotein enhancer increases the effectiveness of albuminpromoter and increases the binding of RNA polymerase complex, therebyproducing more mRNA, ultimately leading to an increase in proteinproduction. The endogenous factors present in liver cells interact withalpha-fetoprotein enhancer region which activates the albumin andalpha-fetoprotein promoters during liver development and in fullydeveloped liver. Because the effect of AFP enhancer is extinguished infully developed liver cells through repression of its activity, theregion associated with repression is not included in our AFP enhancersequence, which allows the enhancer activity to persist in fullydeveloped liver cells.

A suitable form of the AFP enhancer of the present invention isdisclosed in Jin et al., Developmental Biology 336 (2009) 294-300. Aspecific sequence of the AFP enhancer can be found at residues 15-265 ofSEQ ID NO: 1 (TA1 expression cassette).

In another embodiment of the invention, one would use other promoterenhancers suitable for use with a liver-specific promoter. Many normalpromoters are quite large in size and contain multiple regions thatmodulate transcriptional activity as required for the existingphysiological needs at a given time. Therefore, selection of anappropriate promoter enhancer is context dependent. It must work withthe promoter in question. If empirical determinations validatefunctional efficacy of enhancers from other promoters, in conjunctionwith the liver-specific promoter used, appropriate modifications ininsulin expression cassettes can be made to achieve the desired results.Currently, according to the Cold Spring Harbor Laboratory database,there are approximately 400 known regulatory regions and elements thatfunction in liver cells.

Translational Enhancer

By “translational enhancer,” we preferably mean the VEGF translationalenhancer. The Examples below disclose the use of the VEGF translationalenhancer. This element is designed to enhance translation of thefunctionally linked protein encoding sequence. The VEGF translationalenhancer acts as a ribosomal entry site; it increases the effectivenessof the translation process. Thus, its presence causes a larger amount ofinsulin protein production from a given amount of insulin mRNA.

A specific sequence of the VEGF translational enhancer can be found atresidues 707-870 of SEQ ID NO: 1 (TA1 expression cassette) and residues755-918 of SEQ ID NO: 2 (TA4 expression cassette).

Glucose Inducible Regulatory Elements (GIREs)

The vector of the present invention requires 1-5 GIREs, preferably 2-4GIREs, most preferably 3 GIRES. One may find the sequence for suitableGIREs at residues 279-369 in SEQ ID NO: 1 and 46-136 in SEQ ID NO: 2.Suitable GIREs are also described below in the Examples and may also befound at U.S. Pat. Nos. 7,425,443 and 6,933,133, both incorporated byreference.

As used herein, a “glucose inducible regulatory element” (GIRE) refersto a polynucleotide sequence containing at least one pair of perfectCACGTG motifs, each member of the pair separated from the other by asequence of five base pairs. A “glucose responsive regulatory module”contains one or more GIREs. In one example, the regulatory elements wereinserted 5′ of the 5′ untranslated region of the human proinsulin geneand then cloned into an adenovirus vector which was used to transfecthepatocytes. As the Examples below demonstrate, the GIREs providetranscriptional regulation of insulin mRNA in hepatocytes in response tophysiologically relevant glucose concentrations.

Promoter Sequence

The constructs of the present invention also involve the use of a genepromoter, preferably an albumin promoter. The albumin promoter is ahepatocyte (liver) specific promoter and is used to ensure thatproduction of insulin is restricted only to liver cells. Therefore, ifsome of the insulin gene construct ends up in organs other than liver,the construct will not be expressed. Additionally, various componentsand mechanisms necessary to confer glucose responsiveness to insulinexpression using gene constructs of the present invention are endogenousto liver cells. As illustrated in Examples herein, the rat albuminpromoter (184 bp), (Heard et al., Determinant of rat albumin promotertissue specificity analyzed by an improved transient expression system.Mol Cell Biol 1987; 7: 2425) was generated by PCR using rat genomic DNAtemplate, as described previously (Alam et al., Glucose-regulatedinsulin production in hepatocytes. Transplantation 2002; 74:1781). Theuse of the rat albumin promoter sequence in the example is provided forillustrative purposes only. Constructs containing an albumin promoterfrom other species, such as humans, are expected to confer similarproperties to the constructs.

One may obtain an albumin promoter by use of primers and PCRamplification after examination of SEQ ID NOs: 1 and 2. The promotersequence is found at residues 380-706 of SEQ ID NO: 1 and 147-474 of SEQID NO: 2 and 380-707 of SEQ ID NO:3.

In principle, any constitutively active liver cell specific promotercapable of sustained moderate to high level transcription can besubstituted for albumin promoter. An example of such a promoter is alpha1-antitrypsin inhibitor (Hafenrichter D G et al. Blood 1994; 84,3394-404). Currently, according to the Cold Spring Harbor Laboratorydatabase there are approximately 300 known liver specific promoters.

Gene Encoding Insulin with Modified Peptidase Site

The vectors of the present invention comprise a gene encoding insulin,preferably human insulin or non-human mammalian insulin, with a modifiedpeptidase site. The insulin genes of the present invention are alsodisclosed in SEQ ID NO: 1 at residue 871-1203 and SEQ ID NO: 2 atresidue 919-1251.

In one aspect of the invention, one may wish to treat non-human animals.To ensure that no immune reaction to insulin occurs when diabeticanimals, such as cats or dogs, are treated using insulin gene therapy,one would use species-specific insulin in minicircle DNA for treatinganimals. For example, one would use published sequences of insulin forcats and dogs (Kwok et al. 1983 J Biol Chem 258 2357-2363) to generate3′ and 5′ primers to amplify the coding sequence of insulin from cDNApreparations made from isolated pancreatic RNA from respective speciesby standard molecular biology techniques. Alternatively, the codingsequence can also be chemically synthesized. Similarly one may wish tosubstitute insulin sequences from other animals when treating thoseanimals. These sequences are readily available.

Human insulin cDNA was modified at two junctions of proinsulin whereproteolytic processing and maturation of insulin occurs by specificenzymes residing in beta cells but absent in liver cells. Themodification at the B and C peptide of human insulin from KTRR to RTKRand at the C and A peptide from LQKR to RQKR makes the two insulinjunctions compatible with cleavage specificity of endogenous protease,furin, of liver cells. These modifications are described in thefollowing publications: Simonson G D, Groskreutz C M, Gorman C M, et al.Synthesis and processing of genetically modified human proinsulin by ratmyoblast primary cultures. Hum Gene Ther 1996; 7: 71.; Groskreutz D J,Sliwkowski M X, Gorman C M. Genetically engineered pro-insulinconstitutively processed and secreted as mature, active insulin. J BiolChem 1994; 269: 6241.

Regarding the use of non-human insulin, all modifications to thesequence of preproinsulin will be similar in nature to that describedfor human insulin, wherein the recognition/processing sites forpeptidases found in β-cells (and neuroendocrine tissue) will be changedto sites that can be processed by commonly found proteases in liver(such as furin) and other cells. There are some minor sequencedifferences in insulin from various species but the key point is toretain the authentic sequence of mature insulin for the given speciesafter processing.

The purpose of the specific mutation is to change the amino acidsequence in such a way that proteolytic processing is possible bycommonly found furin. There are multiple codons for several of the aminoacids. Theoretically, one can alter the DNA sequence by using analternative codon but still produce the same polypeptide.

These modifications have been successfully used by Applicants in apublished report (Alam T, Sollinger H W. Glucose-regulated insulinproduction in hepatocytes. Transplantation 2002; 74:1781). An unmodifiedinsulin gene will produce unprocessed proinsulin because the specificenzymes necessary for the maturation by proteolytic processing areabsent in liver cells. Proinsulin has minimal biological activity ofapproximately 100 fold less than the mature insulin.

One may obtain a modified insulin gene by use of primers and PCRamplification with knowledge of the insulin gene in SEQ ID NOs: 1, 2 and3.

Albumin 3′ UTR

The albumin 3′ UTR is known to contribute to longevity of the albuminmRNA in hepatocytes. This sequence was obtained from an expressionvector plasmid from Mirus (pMIR0375) but this sequence can also beamplified by PCR using reverse transcribed mRNA from liver. The albumin3′ UTR sequence is disclosed in SEQ ID NO: 1 at 1204-2077 and at SEQ IDNO: 2 at residues 1252-2125.

HGH Intron

Two of the constructs of the present invention, TA4 and TA2, comprisethe HGH intron. The HGH intron is known to add to the efficiency of mRNAprocessing and helps in yielding quantitatively more mRNA. There areseveral other introns, such as beta-globin, that serve similar functionto a varying degree. However, the HGH intron is known to function welland is preferred. The HGH intron may be amplified by PCR from thecommercially available plasmid pAAV-LacZ [Stratagene, La Jolla, Calif.].The sequence can also be readily amplified by PCR using genomic DNA asthe template. The sequence of the HGH intron is disclosed at residues475-754 of SEQ ID NO: 2 and 708-987 of SEQ ID NO:3.

Minicircle Embodiment

Optionally, the vector of the present invention is in the “minicircleDNA” format. This is a vector that is virtually devoid of all DNAsequences that are unrelated to expression of insulin. The originalminicircle DNA production vector was obtained from the laboratory ofMark Kay, described in the following publications: Chen, Z Y, He, C Y,Ehrhardt, A and Kay, M A (2003). Minicircle DNA vectors devoid ofbacterial DNA result in persistent and high-level transgene expressionin vivo. Mol Ther 8: 495-500, and Chen Z Y, He C Y, Kay M A (2005)Improved production and purification of minicircle DNA vector free ofplasmid bacterial sequence and capable of persistent transgeneexpression in vivo. Human Gene Ther, 16:126. A newly revised method toeasily produce minicircle DNA was published recently (Kay M A, He C,Chen Z. A robust system for production of minicircle DNA vectors. 2010.Nature Biotech 28, 1287). The vector and the E. coli needed to producethe minicircle are commercially available from System Biosciences (SBI),Mountain View, Calif. (systembio.com), and are currently used by us.

The present invention of insulin expression constructs conforms to thegenerally accepted and proven placement scheme of various elements inrelation to each other. Thus, the gene expression constructs of thepresent invention are comprised of AFP enhancer-conditionalinducer-promoter-intron 1-gene-intron 2-termination/5′ UTR. In ourExamples, the GIREs are the conditional inducers and there is atranslational enhancer inserted after the HGH intron and before themodified insulin gene. After the insulin gene, the second intron forefficient mRNA processing is from albumin followed by the 3′ UTR ofalbumin.

FIGS. 4, 5 and 10 disclose preferred placement of elements in geneconstructs of the present invention.

Method of the Present Invention

In one aspect, the present invention is a method of controlling bloodglucose levels in a mammalian patient (preferably a human or non-humanmammal), comprising the steps of treating a mammal with hepatocytes thathave been modified with a first, second or third vector, as describedabove. The first vector comprises promoter enhancer, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase site and an albumin 3′ UTR and lacks an HGHintron. The second vector comprises an HGH intron, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase site and the albumin 3′ UTR and lacks promoterenhancer. The third vector comprises an HGH intron, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase site, the albumin 3′ UTR and promoter enhancer.

One would observe the mammal's blood glucose and insulin levels aftervector treatment and note that the mammal's blood glucose or insulinlevels are controlled and normal.

To test our insulin expression constructs ex vivo in hepatocytes, TA1,TA2 and TA4 insulin constructs were cloned in adenovirus vector, asdescribed earlier (Alam T, Sollinger H W. Glucose-regulated insulinproduction in hepatocytes. Transplantation 2002; 74:1781.) Freshlyisolated normal rat hepatocytes were plated on collagen coated cellculture plates and transfected with adenovirus containing the insulingene construct. These cells were then exposed to low (3.5 mM), normal(5.6 mM) and high (27.5 mM) concentrations of glucose. Aliquots ofmedium were drawn at various time intervals and insulin present in theculture medium was quantitated by ELISA. Results showed that hepatocytestransfected with each insulin construct produced insulin in a glucoseconcentration dependent manner (FIG. 9). At the high concentration ofglucose, the amount of insulin production was 4-10× higher than at thelow concentration of glucose.

By “controlled,” we mean that the method of the present invention ispreferably characterized by tight control of glucose regulation. Thetight control refers to the empirical observation of glucose regulationitself. In non-diabetic individuals, the blood glucose returns to normalat 2 hr post meal. Before the present invention, one would haveanticipated that following the correction of hyperglycemia in a mammalin response to elevation in blood glucose levels, the preformed insulinmRNA would remain for a while and continue producing insulin. Dependingon how long such a condition persists, one would expect that the mammalwould then become hypoglycemic. However, our results showed that theinsulin levels in serum increased soon after the increase in bloodglucose levels, as we had anticipated, but the insulin levels did notstay high for too long and followed the blood glucose level curve with adelay of about 15-30 min (See FIG. 8).

Typically, the present invention provides that insulin levels will staywithin 0.5 uU-100 uU/ml. (This is comparable to the maximum amount ofinsulin that is released from normal islets under hyperglycemicchallenge of approximately 100 uU/ml.).

Typically, blood glucose concentration will stay within 80-150 mg/dlafter treatment. The high end relates to a temporary rise soon afterhaving a meal. If glucose concentration does rise above 150, the leveldoes not stay at that level for beyond a short period (30-60 min).

Poorly controlled diabetes causes hyperlipidemia and the severity ofhyperlipidemia is dependent on the degree of hyperglycemia. Liverfunction tests are performed for two reasons. Severe diabetes isassociated with a degree of systemic inflammatory responses, includingelevation in serum levels of some liver enzymes. Our data provideevidence that following insulin gene therapy, a correction in serumlevels of liver enzymes is apparent. Secondly, the hydrodynamic deliverprocedure is known to cause a transient stress to liver but this damageis short-lived. Our data support these findings and assert that there isno long-term risk associated with gene-therapy in the context of liverfunction. In fact, the therapy normalizes the liver function, asevidenced by the albumin production.

In one embodiment of the present invention, the lipid and/or liverenzyme profile of the treated animal is corrected. In one embodiment ofthe present invention, the animal will have a lipid/enzymes panelwherein the plasma lipid or liver enzymes concentration is equivalent toor less than a normal control. By “normal control” we mean an animal whois not diabetic. By “lipid/enzymes panel” we mean either a plasmatriglycerides measurement, an alanine transaminase (AST), an aspartatetransaminase (ALT) or plasma albumin measurement. In another embodimentof the invention, we would expect to see a drop in cholesterol (mg/dl)of at least 20% compared to a diabetic control. Based on a conservativeestimate, approximately 1 week may be sufficient for a substantialcorrection or normalization of hyperlipidemia.

The method of the present invention involves the treatment of a mammal,preferably a human patient or non-human mammal, with the vectors of thepresent invention.

Introduction of our insulin expression constructs into liver cells canbe achieved either in vivo or ex vivo. In the first method, the geneconstruct will be introduced either using a minicircle DNA byhydrodynamic method described herein, injecting the condensed minicircleDNA nanoparticles as such, or after coating nanoparticles with compoundsthat are known to target liver cells. Alternatively, liver cells will beharvested from the patient through a biopsy, expanded in cell culture,and transfected with a construct of the present invention usingminicircle or safe viral vectors such as adeno-associated virus (AAV)(already used in many clinical trials).

The transfected cells will be tested for their ability to produceinsulin and modulate the quantity of production of insulin in responseto changes in concentrations of glucose. Appropriate number of cells toprovide necessary amount of insulin (assisted with information from exvivo measurements) will be transplanted into the liver of the mammal viaradiological and ultrasound guidance. The ex vivo method allows forlower vector load as well as for targeted delivery of gene.

Incorporating scaffold/matrix-attachment regions (S/MARs) that serve asconstitutively active anchors for the nuclear scaffold (Wong S P et al.Gene Ther. 2011; 18(1):82-7; Heng H H et al. J Cell Sci. 2004; 117(Pt7):999-1008; Lufino M M et al., Nucleic Acids Res. 2007; 35(15):e98.PMCID: 1976449) will likely increase the survival of TA1minicircle(TA1m)-containing S/MARs (TA1m-S/MAR) or other minicircles due to theassociation of SARs with the nuclear scaffold and may also reduce genesilencing (Wong S P et al. Gene Ther. 2011; 18(1):82-7).

The Examples below describe the delivery of DNA into rat liver byhydrodynamic procedures (Zhang G. et al., Methods Mol Biol 245:251-264,2004; Zhang G. et al., Hum Gene Ther 8:1763-1772, 1997). The delivery ofthe DNA into mammalian liver will typically not be by the hydrodynamicmethod described in the Examples. Other alternatives, such asnano-particles of condensed DNA that do not require a large volume andhigh pressure, will be employed. In a preferred embodiment, suitableglucose regulation will last at least 2-4 weeks after treatment with theconstructs.

Hydrodynamic venous delivery of naked plasmid DNA has led to successfulgene uptake and transduction of liver cells (Sebestyen M G et al. HumGene Ther. 2007; 18(3):269-85. PMCID: 2268901; Wooddell C I et al. JGene Med. 2008; 10(5):551-63; Wooddell C I et al. Hum Gene Ther. 2011;22(7):889-903. PMCID: 3135275). This method relies on a rapid,high-volume intravenous injection (˜10% of body weight). However, withhydrodynamic venous delivery, much of the DNA is absorbed in the venoussystem, particularly the lungs, prior to its delivery to the liver. Asan alternative, one may inject the vectors into the arterial system,such as the femoral artery, which has a systolic blood pressure ofapproximately 120 mm/Hg. The remaining vector will reach the liver viathe portal vein at 10-12 cm.H₂O. (Blood pressure is usually presented inmm of Mercury (mm/Hg). Venous blood pressure is lower and sometimes aunit based on cm of water (cm/H₂O) is used.) Overall, intra-arterialinjection should greatly enhance TA1m constructs uptake compared tohydrodynamic venous delivery.

Injection into the femoral artery should dramatically increase vectorliver uptake and transduction of hepatocytes. We expect that treatedanimals will maintain normoglycemia for more than 1 month, even when fedad libitum.

EXAMPLES Example 1 Generation of Constructs

Human insulin based gene constructs containing various elements tomodulate expression were generated with the aim of producingbiologically active insulin in response to changes in glucose levels.

Four insulin constructs designated TA1, TA2, TA3, and TA4 containvarious elements in order shown below:

AFP- Alb- HGH- Human Alb Enhancer 3GIREs Promoter intron Insulin 3′-UTRTA1 Y Y Y N Y Y TA2 Y Y Y Y Y Y TA3 N Y Y N Y Y TA4 N Y Y Y Y Y

AFP-Enhancer:

Alphafetoprotein enhancer was used from Mirus vector pMIR0375.

3GIREs:

3 units of glucose inducible regulatory elements are connected intandem; the sequences are based on S14. S14 is a glucose responsivetranscriptional enhancer. The elements responsible for glucose-dependenttranscriptional enhancement have been identified in published work: ShihH. Towle H C. Definition of the carbohydrate response element of the ratS14 gene: Context of the CACGTG motif determines the specificity ofcarbohydrate regulation. J Biol Chem 1994; 269: 9380.

Alb-Promoter:

Albumin promoter (Albumin promoter was amplified by PCR from rat genomicDNA, as described in U.S. Pat. No. 6,352,857, U.S. Pat. No. 6,933,133,U.S. Pat. No. 7,425,443 and the following publication: Alam T, SollingerH W. Glucose-regulated insulin production in hepatocytes.Transplantation 2002; 74:1781)

HGH-Intron:

Human growth hormone intron was amplified by PCR from a commerciallyavailable plasmid pAAV-LacZ (Stratagene).

Human Insulin:

Human insulin cDNA sequence was modified at B-C and C-A junction forfurin cleavage compatibility so that liver cells are able to processpreproinsulin to functional insulin.

Alb 3′-UTR:

Albumin 3′-untranslated region was used from Mirus vector pMIR0375. Itcan also be amplified by PCR using reverse transcribed mRNA from liver.

Insulin expression constructs described above were incorporated intoreplication-defective adenovirus for transient expression for initialtesting purposes. Insulin expression in rat hepatocytes, ex vivo, wasglucose responsive and each construct yielded significantly higheramount of insulin (4-12 fold improvement over the previously describedconstructs), Alam & Sollinger, Transplantation. 2002 Dec. 27;74(12):1781-7.

TA2 and TA3 were also tested in ex vivo insulin production. (See FIG.9).

Initially we incorporated TA1 in an adenovirus vector to test itsability to control hyperglycemia in rats that were rendered diabetic bystreptozotocin (STZ) treatment. Results showed that such a treatmentfully corrected fasting hyperglycemia, restored the weight loss causedby diabetes to normal rate of weight gain and significantly reducedpostprandial hyperglycemia.

The adenovirus vectors containing TA1-TA4 were tested individually fortheir ability to correct diabetic hyperglycemia in STZ-rats. Resultsshowed a full correction of fasting hyperglycemia and a partialcorrection of postprandial hyperglycemia. The benefit of a single genetherapy treatment on overall metabolism and preventing body weight losslasted well beyond the time of full correction of fasting hyperglycemia.During the period of full correction of fasting hyperglycemia, the rateof weight gain in diabetic rats treated with our insulin gene constructswas indistinguishable from the normal controls.

To improve efficacy of gene therapy through better gene expression byincreasing levels and duration of insulin expression, minicircles of DNAcontaining only the gene expression constructs were produced. All of theabove insulin gene expression constructs were cloned into a plasmidvector p2φC31 as previously described (Chen et al., 2003, Mol Ther, 8,495-500; Chen et al., 2005, Human Gene Ther, 16, 126-131). Thispublished method of Chen et al. was substantially modified to improvepurity of minicircle DNA as described below.

Encouraged by results from transient expression afforded by adenovirusvector, we generated a plasmid vector, known as “minicircle DNA,” thatis virtually devoid of all DNA sequences that are unrelated toexpression of insulin. TA1 minicircle was introduced into the livers ofrats via an established hydrodynamic procedure (Zhang G. et al., MethodsMol Biol 245:251-264, 2004; Zhang G. et al., Hum Gene Ther 8:1763-1772,1997). Results obtained from STZ-diabetic rats treated with TA1minicircle DNA show a full correction of hyperglycemia among ad lib fedanimals (FIG. 1) in addition to restoration of rate of weight gain tonormal (FIG. 2) and correction of fasting hyperglycemia.

The TA1-minicircle-treated diabetic rats were subjected to a glucosetolerance test by intraperitoneal injections of 4 gm/kg glucose. Resultsfrom these experiments bore a marked similarity to observations fromnormal control rats. The peak of elevated blood glucose levels appearedat 15 min post injection and hyperglycemia dissipated in about 60 min(FIG. 8). The time to correct hyperglycemia induced by 4 gm/kg glucoseIP injection is similar to normal animals. An increased insulin outputin response to elevated levels of glucose closely follows the rise inglucose levels and insulin production declines as the level of glucoseprogressively reduces.

To confirm that insulin production was glucose-dependent, we measuredhuman insulin levels in plasma at 30 min time intervals and found thatthe human insulin levels peaked at 30 min and declined relativelyquickly, essentially following the blood glucose profile with a 15 mindelay. Thus, there was an approximately 15 min lag between the profilesof blood glucose and insulin levels.

Given the nature of glucose-induced transcription of insulin mRNA thatgives rise to circulating insulin, after achieving euglycemia, continuedpresence of insulin mRNA could have caused a sustained secretion of highlevels of insulin until the mRNA was degraded. Reduction in insulinlevels in only ˜60 min to the levels observed in fasting animals priorto glucose injections, is an unexpected, albeit very desirable, result.

Modifications to the published minicircle DNA production method wereuseful and necessary to obtain pure minicircle DNA that was free ofdetectable unprocessed or partially processed minicircle DNA. Thesemodifications involved elimination of a 2 hr incubation step, claimed tobe necessary for in vivo digestion of the DNA circle that consists ofthe unneeded sequences from the parental plasmid that were eliminatedfrom the minicircle containing the gene of interest. In practice, thisstep was only partially effective.

In our procedure, elimination of this 2 hr incubation step caused noperceptible change in quality or quantity of recovered DNA and the finalproduct was comprised of a mixture of minicircle DNA and the parentalunprocessed plasmid DNA as well as partially processed plasmid DNA. Themixture of DNA thus produced was treated, ex vivo, with a restrictionenzyme that could cut the parental plasmid but not the minicircle DNAcontaining our insulin gene constructs. The product of this reaction waspurified by CsCl equilibrium density gradient to separate the circularDNA from linear DNA.

The TA1 insulin minicircle DNA was tested for its ability to correctdiabetic hyperglycemia in STZ-treated diabetic rats. Groups of rats wererendered diabetic by intravenous streptozotocin injections (100 mg/kg).The TA1 insulin minicircle DNA was injected via tail vein into diabeticrats according to a previously published method (hydrodynamic deliverymethod described by J. Wolff group, Zhang G. et al., Methods Mol Biol245:251-264, 2004; Zhang G. et al., Hum Gene Ther 8:1763-1772, 1997).Four groups of diabetic rats were injected with indicated amounts of TA1minicircle DNA (1.0 μg, 0.75 μg, 0.5 μg, and 0.025 μg per gm bodyweight). Results are shown in FIGS. 1, 2, and 3.

This is the first time we have been able to fully correct blood glucoselevels in diabetic rats fed ad libitum (FIG. 2) by insulin gene therapy.This treatment fully restored rate of weight gain in diabetic rats (FIG.3).

Example 2 Creation of Adenovirus Constructs

Referring to FIG. 12, insulin constructs TA1 and TA4 were created inadenovirus and equal plaque forming units injected into rat livers, asindicated. TA1 and TA4 were used under identical conditions. Both wereable to fully correct fasting hyperglycemia, as shown in FIG. 12.However, the vectors did not fully correct blood glucose levels in ratsfed ad libitum.

Referring to FIG. 13, TA1 in adenovirus was injected into rat livers asindicated. Note that the TA1 treated rats regained the weight initiallylost due to diabetes and maintained a higher weight than the STZdiabetic rats.

Referring to FIG. 14, TA1 or TA4 in adenovirus was injected into liversof diabetic rat groups, as indicated. Note that both TA1 treated and TA4treated rats had maintained higher body weight than STZ diabetic rats.FIG. 14 shows a subset of data points from FIG. 13, representing 0-48days. The information on rate of weight gain in various groups of rats(shown in the inset box on the top left corner) is derived from dataobtained 24 days post treatment (d8-d31). The rate of weight gain indiabetic rats treated with TA1 in adenovirus is equal to normal controlrats, whereas the diabetic untreated rats experienced a net loss ofweight on a daily basis. FIG. 13 has many more data points, and theearly days occupy only a small portion of the area, and therefore thedegree of correction in weight gain may be somewhat difficult toappreciate. FIG. 14 shows this aspect more clearly.

Example 3 Evaluation of Human Insulin in Serum of Diabetic Rats

Referring to FIG. 15, a comparison of human insulin in rat serum showsthat animals treated with TA1 minicircle DNA produce larger amounts ofinsulin compared to other plasmid vectors (pTED110, TA1/pENTR). Theplasmid pENTR contains an ampicillin resistance gene which has beenreplaced by a Kanamycin resistance gene in pTED110, and S/MAR has alsobeen added to it; both have the TA1 insulin construct. The TA1minicircle containing S/MAR produced the most insulin in vivo. TheUltrasensitive Human Insulin ELISA (Mercodia, Inc) has a detection limitof 0.15-20 mU/L, as advertised. The table in FIG. 15 shows the relativein vivo effectiveness of various vectors used for insulin gene therapy.All insulin vectors contained the TA1 expression cassette. pENTR is acommercial plasmid containing an ampicillin resistance gene. The pTED isour modified plasmid where we replaced the ampicillin resistance genewith kanamycin resistance gene to increase the in vivo survival of thevector in non-dividing cells.

We also added S/MAR to pTED110 to increase survival of vector individing cells and to some degree, increase the overall expression. Thedata are in agreement with our vector design expectations.

Finally, when extraneous sequences were eliminated and insulin geneconstructs were used as minicircle DNA molecules, the expression levelsof insulin were significantly increased, more so when S/MAR was includedin the minicircle. In all four sets of experiments, the molarequivalence of TA1 was maintained at a constant level.

The table (FIG. 15) in conjunction with data obtained from adenovirusmediated transduction ex vivo (FIG. 9) and in vivo (FIG. 12 and FIG. 13,FIG. 14, and FIG. 17) provides the proof that the insulin expressioncassettes are able to produce glucose-dependent insulin, as intended.However, the magnitude and longevity of insulin expression does dependon the vector employed to deliver the insulin expression cassette(s). Weexpect that these insulin expression cassettes will be readily adaptableto take advantage of new vectors developed to have bettercharacteristics for gene therapy, such as ease of delivery, expressionlevel, and longevity of expression

Example 4 Second Treatment with TA1M

Referring to FIG. 16, a second treatment with TA1 minicircle DNAcorrects the gradually elevating fasting blood glucose to normal level.

FIG. 1 discloses data that are relevant to the information presented inFIG. 16. As the information in two figures comes from different studies,individual data points are not identical, but the trend is the same. Oneshould refer to the curve corresponding to use of 1.0 μg/gm TA1minicircle DNA in FIG. 1 for comparison. FIG. 16 shows that it appearspossible to correct hyperglycemia by a second treatment as the effect ofthe first treatment diminishes.

Example 5 Hepatocytes Derived from Human Stem Cells

Referring to FIG. 17, hepatocytes were derived from human embryonic stem(hES) cells and human adult induced pluripotent (iPS) cells as describedin Si-Tayeb et al 2010. Hepatology 51(1):297-305. PMCID: 2946078 andwere transduced with TA1 in adenovirus, ex vivo. Three cell cultureplates were kept in medium with low glucose (3.5 mM), and three plateswere kept in high glucose (27.5 mM). Freshly prepared normal rathepatocytes were treated similarly. Flasks containing stem cell derivedhepatocytes were confluent with cells, whereas primary rat hepatocyteplates were ˜60% confluent.

Results (FIG. 17) show a robust production of glucose-dependent insulinin embryonic and induced pluripotent-cell-derived hepatocytes.

Example 6 Examination of Rat Weight after Treatment with MinicircleVectors

Referring to FIG. 18, groups of diabetic rats (minimum number of rats ina group=5) were injected with the indicated insulin minicircle DNA, TA1,TA2, or TA3, via tail veins. Fasting body weights (Mean±S.D) of rats areshown. Normalization in rate of weight gain is similar when TA1m or TA2mwas used, whereas TA3m was less effective.

Example 7 Evaluation of Lipid Profiles

Two groups of rats were rendered diabetic by intravenous streptozotocintreatment. One group of diabetic rats (n=5) was treated with 1 μg TA1minicircle DNA/gm body weight of animal. The second group of diabeticrats was used as an untreated control. A third group of normal rats wasincluded as age matched healthy controls. Blood was drawn from eachexperimental animal after 10 days, and plasma was analyzed for lipidcontents and various markers of liver damage and hepatic function, asshown in the table below:

Aspartate Alanine Alkaline Plasma Animal Triglycerides CholesterolTransaminase Transaminase Posphatase Albumin Groups (mg/dl) (mg/dl)(U/L) (U/L) (U/L) (g/dl) TA1m 53 ± 34 141 ± 15 302 ± 33   77 ± 40 210 ±95 3.4 ± 0.2 Treated Diabetic 704 ± 313 191 ± 36 617 ± 349 152 ± 75 423± 73 2.5 ± 0.2 Normal 100 ± 14  129 ± 6  504 ± 100 106 ± 16 172 ± 55 3.4± 0.1 Control

TA1m treatment corrected all deficiencies caused by the uncontrolleddiabetes. Thus, the levels of cholesterol and triglyceride in plasma oftreated rats were reversed to normal levels. Likewise liver functionmarkers showed an improvement and reduced levels of albumin in diabeticrats returned to normal.

1. A method for obtaining glucose-regulated expression of insulin exvivo in hepatocyte cells, wherein the method comprises delivering afirst, second or third genetic vector for glucose-regulated synthesis ofinsulin into an isolated hepatocyte cell, wherein the first vectorcomprises a promoter enhancer, 1-5 glucose inducible regulatoryelements, a liver-specific promoter, a gene encoding insulin withmodified peptidase sites and an albumin 3′UTR and lacks an HGH intron,wherein the second vector comprises an HGH intron, glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase sites and an albumin 3′UTR and lacks a promoterenhancer, wherein the third vector comprises an HGH intron, glucoseinducible regulatory elements, a liver-specific promoter, a geneencoding insulin with modified peptidase sites, an albumin 3′UTR and apromoter enhancer, and wherein glucose-regulated expression of insulinoccurs.
 2. The method of claim 1 additionally comprising the step oftransplanting the hepatocytes back into a mammal.
 3. The method of claim1, wherein the genetic vector is delivered by exposing the cells to avirus infective for the cells, wherein the virus comprises the geneticconstruct, and whereby at least a portion of the cells are infected bythe virus under suitable conditions and at a sufficient multiplicity. 4.The method of claim 1 wherein the mammal is human.
 5. The method ofclaim 1 wherein the insulin is human insulin.
 6. A vector suitable forcontrolling blood glucose levels in a mammal, wherein the vectorcomprises a promoter enhancer, glucose inducible regulatory elements, aliver-specific promoter, a gene encoding insulin with modified peptidasesites and an albumin 3′UTR and lacks an HGH intron or wherein the vectorcomprises an HGH intron, glucose inducible regulatory elements, aliver-specific promoter, a gene encoding insulin with modified peptidasesites and an albumin 3′UTR and lacks a promoter enhancer or wherein thevector comprises an HGH intron, glucose inducible regulatory elements, aliver-specific promoter, a gene encoding human insulin with modifiedpeptidase sites, an albumin 3′UTR and a promoter enhancer.
 7. The vectorof claim 6 wherein additionally comprising a VEGF-Enhancer.
 8. Thevector of claim 6 wherein the promoter is an albumin promoter.
 9. Thevector of claim 6 wherein the insulin is human insulin.
 10. A method ofcontrolling blood glucose levels in a mammal, comprising the steps of:treating a mammal with a first, second or third vector, wherein thefirst vector comprises a promoter enhancer, 1-5 glucose inducibleregulatory elements, a liver-specific promoter, a gene encoding insulinwith modified peptidase sites and an albumin 3′UTR and lacks an HGHintron and wherein the second vector comprises an HGH intron, glucoseinducible regulatory elements, a liver-specific promoter, a geneencoding insulin with modified peptidase sites and an albumin 3′UTR andlacks a promoter enhancer, wherein the third vector comprises an HGHintron, glucose inducible regulatory elements, a liver-specificpromoter, a gene encoding insulin with modified peptidase sites and analbumin 3′UTR and a promoter enhancer, and observing the mammal'sinsulin levels, wherein the mammal's insulin levels are controlled. 11.The method of claim 1 wherein the vector is in a minicircle format. 12.The method of claim 10 wherein the vector is in a minicircle format. 13.The method of claim 10 wherein the mammal is human.
 14. The method ofclaim 13 wherein the insulin is human insulin.
 15. The method of claim10 wherein the mammal's cholesterol level decreases after treatment. 16.The method of claim 10 wherein the mammal's triglyceride level decreasesafter treatment.
 17. The method of claim 10 wherein the mammal is a cat.18. The method of claim 10 wherein the mammal is a dog.
 19. The methodof claim 10 wherein the mammal is selected for the group consisting ofhamsters, gerbils, rats, mice, rabbits, guinea pigs, chinchillas andferrets.
 20. The method of claim 10 wherein the mammal is a non-humanmammal.
 21. The method of claim 10 wherein the patient has a decrease inthe plasma level of a compound selected from the group of AST, ALT, andalkaline phosphatase after treatment.